(PLA2R1) Sequence Variants in Idiopathic Membranous Nephropathy

CLINICAL RESEARCH www.jasn.org Phospholipase A2 Receptor (PLA2R1) Sequence Variants in Idiopathic Membranous Nephropathy † ‡ Marieke J.H. Coenen,* Julia M. Hofstra, Hanna Debiec, Horia C. Stanescu,§ | Alan J. Medlar,§ Bénédicte Stengel, ¶ Anne Boland-Augé,** Johanne M. Groothuismink,* †† ‡‡ Detlef Bockenhauer,§ Steve H. Powis,§ Peter W. Mathieson, Paul E. Brenchley, † ‡ Robert Kleta,§ Jack F.M. Wetzels, and Pierre Ronco *Department of Human Genetics and †Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; ‡Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie Univ-Paris, Assistance Publique-Hôpitaux de Paris, Department of Nephrology, Tenon Hospital, Paris, France; §Centre for Nephrology, University College London, London, United Kingdom; |Institut National de la Santé et de la Recherche Médicale U1018, CESP, Team 10, Villejuif, France; ¶Université Paris-Sud, Villejuif, France; **Centre National de Génotypage, Institut de Génomique CEA, Evry, France; ††Academic Renal Unit, University of Bristol, Bristol, United Kingdom; and ‡‡School of Biomedicine, University of Manchester, Manchester, United Kingdom ABSTRACT The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membranous nephropathy (iMN). Our recent genome-wide association study showed that genetic variants in an HLA- DQA1 and phospholipase A2 receptor (PLA2R1) allele associate most significantly with biopsy-proven iMN, suggesting that rare genetic variants within the coding region of the PLA2R1 gene may contribute to antibody formation. Here, we sequenced PLA2R1 in a cohort of 95 white patients with biopsy-proven iMN and assessed all 30 exons of PLA2R1, including canonical (GT-AG) splice sites, by Sanger sequencing. Sixty patients had anti-PLA2R1 in serum or detectable PLA2R1 antigen in kidney tissue. We identified 18 sequence variants, comprising 2 not previously described, 7 reported as rare variants (,1%) in the Single Nucleotide Polymorphism Database or the 1000 Genomes project, and 9 known to be common polymorphisms. Although we confirmed significant associations among 6 of the identified common variants and iMN, only 9 patients had the private or rare variants, and only 4 of these patients were among the 60 who were PLA2R positive. In conclusion, rare variants in the coding sequence of PLA2R1, including splice sites, are unlikely to explain the pathogenesis of iMN. J Am Soc Nephrol 24: 677–683, 2013. doi: 10.1681/ASN.2012070730 Idiopathic membranous nephropathy (iMN) is the established that 70% of white patients with active leading cause of nephrotic syndrome in the adult disease have antibodies against PLA2R1.1,3,4 white population. It is characterized by immune complex deposition in the subepithelial layer of the glomerular basement membrane. On the basis of Received July 25, 2012. Accepted December 5, 2012. studies in Heymann nephritis, a model of MN in the M.J.H.C., J.M.H., and H.D. contributed equally to this work; R.K., rat, it was suggested that immune complexes were J.F.M.W., and P.R. contributed equally to this work formed locally, by the binding of antibodies to a Published online ahead of print. Publication date available at 1 podocytic antigen. The description of neonatal www.jasn.org. MN caused by maternal antibodies against neutral Correspondence: Dr. Marieke Coenen, Department of Human 2 endopeptidase provided proof of concept. Further Genetics, Radboud University Nijmegen Medical Centre, PO Box evidence was provided by the discovery of the 9101, 6500 HB Nijmegen, The Netherlands. Email: m.coenen@ M-type receptor for phospholipase A2 (PLA2R1) as gen.umcn.nl the predominant autoantigen in iMN.3 It is well Copyright © 2013 by the American Society of Nephrology J Am Soc Nephrol 24: 677–683, 2013 ISSN : 1046-6673/2404-677 677 CLINICAL RESEARCH www.jasn.org Although no studies have firmly proven that the antibodies are these rare variants (rs141800672, rs149256089, rs150221555, indeed pathogenic, genetics studies have supported the impor- rs140427239, rs149960520, rs149133741, and rs181959329) tant role of PLA2R1 in the pathogenesis of iMN. Recently, we have been reported in publicly available data sets with very have shown highly significant associations to iMN with single low minor allele frequencies (#0.2%). We identified two nucleotide polymorphisms (SNPs) located on chromosomes 6 novel missense variants that have not been previously reported, and 2, by means of genome-wide association studies (GWAS) which result in amino acid changes (c.1160G.A, p.Arg387His; in three independent cohorts.5 The region of interest defined c.2060T.C, p.Leu687Pro). The novel variant c.1160G.A, on chromosome 2 includes the gene PLA2R1. SNPs in this gene p.Arg387His and the splice site change c.3850+1G.A (IVS26 have also been shown to be associated to iMN susceptibility in +1G.A) (rs181959329) were carried by the same patient. two candidate gene association studies performed in Korea and Additionally, nine common SNPs were observed; six of them Taiwan.6,7 These findings together suggest an important role for in the coding regions (rs4665143, rs3749117, rs35771982, PLA2R1 in the pathogenicity of iMN. rs33985939, rs72954858, and rs3828323) and three in noncod- Our GWAS was based on common SNPs, as is typical for ing regions (rs2715918, rs925409, and rs3749119). most GWAS. These SNPs serve as genetic markers used to In the subgroup of 60 anti-PLA2R-positive patients, four identify alleles or haplotypes of interest. Of note, our GWAS of the rare variants (rs149256089, rs140427239, rs149960520, study showed that both the HLA locus on chromosome 6 and and rs149133741) were each observed once in four different the PLA2R1 locus on chromosome 2 were significantly asso- patients. The two novel variants were not observed in this ciated with iMN not only on the SNP level (basic allele test) subgroup (Table 2). but also on the haplotype level (haplotype association test).5 Of the 15 observed coding variants, 12 (rs149133741, These SNPs demarcate alleles that are different in cases versus rs149960520, rs140427239, rs33985939, rs150221555, controls. Not only common SNPs but also rare mutations can rs35771982, rs3749117, rs149256089, rs141800672, rs4665143, be the cause of a signal identified by GWAS.8 Although iMN is and the novel p.Arg387His and p.Leu687Pro) are located with- not a simple genetic disorder with classic Mendelian inheri- in the regions coding for known domains of PLA2R1, of which 1 tance, the strong and significant association of this phenotype (rs3749117) is located within the WMGL motif of the C-type to PLA2R1 (and the HLA locus) leads to the hypothesis that lectin-like domain (CTLD)-1 of PLA2R1. Two other coding rare genetic variants within the coding region of the PLA2R1 variants, one common (rs33985939, p.Arg404His) and one rare gene may explain antibody formation. In the current study, we (rs140427239, p.Tyr499Cys), are situated one and two amino performed sequencing of the coding regions of the PLA2R1 acids, respectively, away from the cysteine residues involved in gene to confirm this hypothesis. disulfide bond formation (C4 and C1 in CTLD2; see Table 1 and Figure 1). The essential splice site at the border between exon 26 and RESULTS intron 26 is altered in one patient, c.3850+1G.A(IVS26 +1G.A) (rs181959329), predicted to lead to exon 26 being Patient Characteristics skipped, which is in frame. This leads to a shortened PLA2R1 Ninety-five patients with biopsy-proven iMN were included: protein missing 46 amino acids within CTLD8, obliterating 48 from the French cohort and 47 from the Dutch cohort, the first cysteine residue involved in disulfide bond formation respectively. Patients were mostly male (75%), and the mean (C1). Of note, this is a known sequence variant with a yet un- age 6 SD at diagnosis was 51615 years. All patients were of determined allele frequency in the general population. self-reported white ancestry. Of note, Single Nucleotide Polymorphism Database release Serum samples were available for 82 patients; anti-PLA2R 136 (February 2012) currently has 138 human sequence variants antibodies were present in 43 samples. In addition, 17 patients curated within the coding region, including splice sites of were considered positive according to the presence of PLA2R PLA2R1. Actually, only 8 of the 138 variants are frequently ob- antigenintheirkidneybiopsyspecimen(SupplementalTableS1). served, nonsynonymous, coding variants. Minor allele frequencies were not established for most of those SNPs. Sequencing All 30 exons, including essential splice sites (defined as the two Association intronic nucleotides—canonical GT and AG—at the intron- The common SNPs identified by our sequencing permit us to exon boundaries, respectively) were sequenced by Sanger tech- analyze the PLA2R1 region in more detail compared with the nology. We identified 18 variants, including 3 in noncoding GWAS that mainly included common intronic SNPs (typical regions (Table 1). for SNP chips used for GWAS). Of the 18 variants observed, 9 were rare or novel (see the In the 95 patients with iMN studied, the allele frequency of Concise Methods section for definitions). These 9 variants several SNPs was significantly different from that of the ethni- were all encountered in a heterozygous state in one patient cally matched controls from the 1000 Genomes project (see each, except for the rare variant rs149133741 (c.2038G.T, Table 1). Specifically, there was a significant difference for

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